Clinically, these findings not only deepen the understanding of m6A-mediated posttranscriptional regulation in CRC but also identify this axis as a promising therapeutic target for overcoming ferroptosis resistance and improving patient outcomes.
Abstract
Background
Extensive clinical evidence has identified metastasis-associated colon cancer 1 (MACC1) as a pivotal cancer-promoting gene that actively fuels the advancement of neoplasms. However, the upstream transcriptional regulators of MACC1 and the specific posttranscriptional mechanisms involving N6-methyladenosine (m6A) modification that govern its expression remain largely undefined. This study aims to elucidate the regulatory network controlling MACC1 expression and its impact on colorectal cancer (CRC) progression.
Methods
MACC1 expression and its potential regulators were systematically analyzed using public databases, including GEPIA, TCGA, and TIMER2, alongside clinical tissue samples and cell lines (SW480, HCT-116, and SW620). Functional experiments were conducted to assess cell viability, proliferation, invasion, and ferroptosis. These methodological approaches encompassed chromatin immunoprecipitation (ChIP), RNA immunoprecipitation (RIP), methylated RNA immunoprecipitation (MeRIP), as well as dual-luciferase reporter systems. Furthermore, in vivo validation was performed using a nude mouse xenograft model.
Results
MACC1 was significantly upregulated in CRC tissues and cell lines, and its high expression correlated with an unfavorable prognosis. Functional assays revealed that silencing MACC1 inhibited CRC cell proliferation and invasion while inducing ferroptosis. Mechanistically, RNA binding protein 15 (RBM15) was identified as a key m6A methyltransferase component that stabilized MACC1 mRNA in an insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1)- dependent manner. Furthermore, zinc finger and BTB domain-containing 33 (ZBTB33) was found to transcriptionally activate RBM15 by binding to its promoter region. Knockdown of RBM15 inhibited CRC cell invasion and proliferation and induced ferroptosis; these effects were notably reversed by MACC1 overexpression. Moreover, ZBTB33 silencing inhibited the key malignant phenotypes of CRC cells and induced ferroptosis by regulating RBM15. Further, RBM15 depletion suppressed tumor growth, which was attenuated by the restoration of MACC1.
Discussion
Our study unveils a novel ZBTB33/RBM15/MACC1 signaling axis that drives CRC progression. Clinically, these findings not only deepen the understanding of m6A-mediated posttranscriptional regulation in CRC but also identify this axis as a promising therapeutic target for overcoming ferroptosis resistance and improving patient outcomes.
Conclusion
These findings uncover a novel ZBTB33/RBM15/MACC1 regulatory axis in CRC, where ZBTB33 transcriptionally activates RBM15 to enhance MACC1 mRNA stability, ultimately suppressing ferroptosis and promoting tumor progression.
Lung adenocarcinoma (LUAD), the most common subtype of lung cancer, is associated with substantial global mortality. Nuclear receptor coactivator 5 (NCOA5) has been implicated in several malignancies; however, its functional role and regulatory mechanisms in LUAD remain largely unknown. In this study, NCOA5 expression was evaluated in 94 paired LUAD and adjacent tissues using immunohistochemistry, RT-PCR, and Western blotting. Functional analyses were conducted using shRNA knockdown, CRISPR-mediated knockout, and overexpression models to assess the effects of NCOA5 on LUAD cell proliferation, migration, invasion, apoptosis, cell cycle progression, and organoid formation. Xenograft models were used to validate tumorigenicity in vivo. IP-MS and co-immunoprecipitation identified NCOA5-interacting proteins, while ChIP-seq and dual-luciferase assays interrogated downstream transcriptional regulation. NCOA5 was significantly upregulated in LUAD tissues and associated with advanced stage, poor differentiation, and reduced overall survival, serving as an independent prognostic factor (
P
< .040). NCOA5 knockdown inhibited LUAD cell proliferation, migration, invasion, induced G0/G1 arrest, promoted apoptosis, reduced organoid formation, and suppressed xenograft growth, whereas NCOA5 overexpression produced the opposite effects. Among 39 candidate interacting proteins identified by IP-MS, ZCCHC3 was validated as a direct NCOA5-binding partner. ZCCHC3 depletion phenocopied NCOA5 loss and reversed NCOA5-induced proliferation, migration, invasion, and colony formation, supporting a functional NCOA5–ZCCHC3 interaction. ChIP-seq analysis identified fibroblast growth factor 22 (FGF22) as a direct transcriptional target of NCOA5. FGF22 was markedly downregulated in LUAD tissues and higher FGF22 expression was associated with improved patient survival. NCOA5 suppressed FGF22 transcription, while NCOA5 inhibition increased FGF22 expression. Functionally, FGF22 knockdown enhanced LUAD aggressiveness, whereas FGF22 restoration abrogated the oncogenic effects of NCOA5 in vitro and in vivo. Collectively, these findings identify a previously unrecognized NCOA5–ZCCHC3/FGF22 axis that drives LUAD progression and provide new mechanistic insight into the role of NCOA5 in LUAD biology.
Yiran Yu, Wen Jin, Erdun Chaogetu et al.· Cell Death & Disease· 0 citations
BACKGROUND
Colorectal cancer peritoneal metastasis (CRCPM) represents a highly aggressive clinical condition associated with limited therapeutic options and poor prognosis. Anoikis resistance, a specialized form of apoptosis induced by loss of cell-matrix interactions, is essential for tumor cell survival during peritoneal dissemination. However, the molecular mechanisms underlying anoikis resistance in CRCPM remain poorly understood.
METHODS
Differentially expressed lncRNAs associated with CRCPM were identified using microarray analysis and validated in clinical specimens. Gain- and loss-of-function assays were performed to evaluate the biological role of MIAT in colorectal cancer (CRC) cells, patient-derived organoids, and in vivo models. RNA pull-down, luciferase reporter, and RNA immunoprecipitation (RIP) assays were conducted to investigate the ceRNA mechanism. Methylated RNA immunoprecipitation (MeRIP) and RNA stability assays were used to assess m6A modification and its regulatory effects on MIAT.
RESULTS
MIAT was significantly upregulated in CRCPM tissues and anoikis-resistant CRC cells and was associated with poor prognosis. Functional assays demonstrated that MIAT silencing suppressed CRC cell proliferation, migration, invasion, and anoikis resistance in vitro and inhibited tumor growth and peritoneal metastasis in vivo. Mechanistically, MIAT acted as a competing endogenous RNA by sponging miR-181a-5p, thereby upregulating the anti-apoptotic protein MCL1. Furthermore, MIAT stability was enhanced by METTL3-mediated m6A modification in an IGF2BP2-dependent manner.
CONCLUSIONS
These findings identify a novel m6A-MIAT/miR-181a-5p/MCL1 signaling axis that promotes anoikis resistance and peritoneal metastasis in CRC. This study provides mechanistic insights into CRCPM progression and highlights MIAT as a potential prognostic biomarker and therapeutic target.
Lin Shu, Liu Yang, Yilin Yin et al.· International Immunopharmaco...· 0 citations
BACKGROUND
Osteosarcoma (OS) represents a common primary malignant bone tumor associated with unfavorable clinical outcomes. Growing evidence underscores the crucial involvement of N6-methyladenosine (m6A) modifications in tumor development, but the specific mechanisms underlying the m6A regulatory network in OS remain to be elucidated.
METHODS
Potential key target genes in OS were identified through bioinformatic analyses, followed by the characterization of m6A-related regulatory proteins, specifically, writer and reader proteins, which showed significant associations with these targets. To elucidate the mechanistic role of m6A methylation in regulating UHRF1 expression, a series of in vitro assays were conducted. These included RNA pull-down, MeRIP-PCR, dot blot, dual-luciferase reporter assays, and RNA stability assays, which collectively confirmed the interaction between m6A regulatory proteins and UHRF1 mRNA. For functional investigations, OS cell lines (U2OS, Saos2, and 143B) with gene silencing or overexpression were established, and the role of UHRF1 in cellular proliferation, migration, and invasion was assessed using CCK-8 assays, Transwell migration and invasion assays, flow cytometry, and wound healing assays. In addition, GSH/GSSG ratio, Fe2+ concentration, and ROS levels were measured using commercial assay kits to explore ferroptosis-related functional mechanisms. To validate the in vivo relevance of our findings, a xenograft mouse model was established. Finally, functional rescue experiments were performed to mechanistically confirm the critical role of the ZCCHC4-UHRF1-CDO1 regulatory axis in OS progression.
RESULTS
ZCCHC4, functioning as an m6A methyltransferase, enhances the stability of UHRF1 mRNA by catalyzing its m6A modification, thereby promoting increased expression of UHRF1. In parallel, IGF2BP3, an established m6A reader protein, specifically recognizes and binds to the m6A-modified sites on UHRF1 mRNA, further stabilizing the transcript and modulating its downstream biological functions. METTL3/METTL14 knockdown experiments ruled out the contribution of classical m6A methyltransferases, confirming that ZCCHC4 is the primary methyltransferase for UHRF1. Dual-luciferase assays and bisulfite sequencing revealed that UHRF1 suppresses CDO1 transcription by inducing high methylation of its promoter, thereby reducing ROS/Fe2+ levels and increasing GSH, which in turn blocks ferroptosis. In U2OS, Saos2, and 143B cells, silencing UHRF1 or ZCCHC4 inhibited proliferation, migration, and invasion while activating ferroptosis. Overexpression of UHRF1 had the opposite effect. In vivo models confirmed that UHRF1 silencing inhibited tumor growth. Furthermore, UHRF1 overexpression partially reversed the phenotypes induced by ZCCHC4 knockdown.
CONCLUSION
In summary, this study reveals for the first time the complete molecular mechanism by which ZCCHC4-mediated UHRF1 m6A methylation promotes OS progression through epigenetic suppression of CDO1 transcription and inhibition of ferroptosis. This regulatory axis (ZCCHC4-IGF2BP3-UHRF1-CDO1-ferroptosis) provides multiple therapeutic targets for OS and lays a solid foundation for the future development of anticancer strategies based on the regulation of ferroptosis, while also offering a promising pathway for clinical translation.
Kai Song, Ju Liu, Bowen Han et al.· Bone· 0 citations
Background Gastric cancer (GC) remains a major cause of cancer-related mortality, highlighting the need to clarify the stromal and post-translational mechanisms underlying its progression. Methods We integrated single-cell RNA sequencing, independent transcriptomic cohorts, clinical tissue analyses, functional assays, co-immunoprecipitation (Co-IP), multiple immunofluorescence labeling, and a xenograft model to investigate the cellular distribution, biological function, and mechanisms of regulation for matrix metalloproteinase 2 (MMP2) in GC. Results Single-cell analysis of paired tumor and adjacent normal tissues suggested an increased relative abundance of fibroblast populations and higher extracellular matrix-associated activity in GC tissues. MMP2 was preferentially expressed in cancer-associated fibroblasts, particularly in transcriptionally defined universal fibroblast-like and myofibroblastic CAF states. Trajectory analysis further suggested that the MMP2-high universal fibroblast-like population occupies an early and potentially transitional position along the fibroblast-state continuum. These findings were supported by an independent single-cell dataset, TCGA-STAD analysis, and tissue staining. In CAF–gastric cancer cell co-culture assays, CAFs enhanced tumor-cell migration and invasion, while modulation of the FBXW7–MMP2 axis altered these phenotypes. MMP2 overexpression promoted GC cell proliferation, migration, and invasion, whereas MMP2 silencing had the opposite effects. Mechanistically, FBXW7 interacted with the intracellular pool of MMP2, enhanced its K48-linked polyubiquitination, and reduced MMP2 protein stability through a proteasome-dependent process. FBXW7 overexpression attenuated MMP2-associated malignant phenotypes in vitro and restrained MMP2-driven tumor growth in vivo. Clinical tissue analyses further showed an inverse association between FBXW7 and MMP2 expression, while elevated MMP2 was associated with unfavorable survival. Conclusion Our findings identify MMP2-high fibroblast states as an important stromal feature of GC and reveal FBXW7-associated regulation of intracellular MMP2 stability as a potential therapeutic vulnerability.
Changjiang Hao, Shengping Jiang, Songpu Wu et al.· Frontiers in Immunology· 0 citations
Cervical cancer remains the fourth most common malignancy among women worldwide, and patients with advanced-stage disease continue to experience poor clinical outcomes despite the availability of targeted therapies. In this study, we investigated the epigenetic role of endothelial cell-specific molecule 1 (ESM1), a soluble proteoglycan, and established an oncogene whose regulatory mechanisms in cervical cancer remain largely unexplored. The epigenetic mechanisms underlying tumor progression remain incompletely understood. Here, we identify ESM1 as a critical epigenetic regulator of cervical cancer malignancy. Integrative analyses of public datasets and clinical specimens revealed that marked ESM1 overexpression correlated with adverse patient prognosis. Functional loss- and gain-of-function studies have demonstrated that ESM1 is essential for maintaining proliferative, clonogenic, migratory, and invasive phenotypes in cervical cancer cells. Transcriptomic profiling revealed that inhibitor of DNA binding 3 (ID3) is a direct downstream tumor suppressor repressed by ESM1. Mechanistically, ESM1 selectively upregulates DNA methyltransferase 3 A (DNMT3A) to induce promoter hypermethylation and transcriptional silencing of ID3. Pharmacological demethylation reactivates ID3 expression and attenuates metastatic capacity. In vivo xenograft and experimental metastasis models validated that ESM1 depletion significantly impaired tumor growth and lung metastasis while increasing ID3 expression. These findings identify the ESM1/DNMT3A/ID3 axis as a novel epigenetic driver of cervical cancer and a potential therapeutic target.
Chen-Lin Yu, Chia-Liang Lin, Hsiang-Lin Lee et al.· Cell Death Discovery· 0 citations
BACKGROUND
Breast cancer (BC), the most widespread malignancy in women globally, is characterized by complex and heterogeneous mechanisms. Lectin, mannose binding 2 (LMAN2), a potential oncogene highly expressed in BC, is linked to poor prognosis. The transcription factor forkhead box protein A1 (FOXA1) is often overexpressed in BC and tied to malignant phenotypes, but how it regulates LMAN2 to influence BC progression remains unclear.
METHODS
LMAN2 expression in BC and its association with patient prognosis were analyzed via the GEPIA database. mRNA and protein levels were detected by qRT-PCR and Western blot. Cell proliferation, angiogenesis, invasion/migration, and apoptosis were evaluated using EdU, tube formation assay, Transwell, and flow cytometry. The ferroptosis status of cells was assessed by detecting ferroptosis-related indicators. The transcriptional regulatory effect of FOXA1 on LMAN2 was verified using JASPAR database, chromatin immunoprecipitation (ChIP), and dual-luciferase reporter assays. The in vivo effect of the FOXA1/LMAN2 axis on BC growth was validated using a nude mouse xenograft model, and related protein expression was detected by immunohistochemistry (IHC).
RESULTS
LMAN2 was upregulated in BC, with high expression linked to poor survival. Silencing LMAN2 was associated with reduced BC cell proliferation, migration, invasion, and angiogenesis, concomitant with increased ferroptosis and apoptosis. FOXA1 binding sites were identified in the LMAN2 promoter; FOXA1 directly bound to this region to enhance LMAN2 transcription, and FOXA1 knockdown reduced LMAN2 expression. FOXA1 overexpression enhanced BC cell malignancy and suppressed ferroptosis, with these effects reversed by concurrent LMAN2 silencing. In vivo, FOXA1 knockdown restrained xenograft tumor growth, and this effect was reversed by concurrent LMAN2 overexpression.
CONCLUSION
FOXA1 bound to the LMAN2 promoter to enhance its transcription, thereby promoting BC progression at least partially through ferroptosis suppression and malignant phenotype induction.
Zhi-Tao Zhang, Shaoyu Wang, Haifeng Sun et al.· Tissue & Cell· 0 citations
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